RNA-Dependent Cysteine Biosynthesis in Bacteria and Archaea

Abstract

The diversity of the genetic code systems used by microbes on earth is yet to be elucidated. It is known that certain methanogenic archaea employ an alternative system for cysteine (Cys) biosynthesis and encoding; tRNA^Cys is first acylated with phosphoserine (Sep) by O-phosphoseryl-tRNA synthetase (SepRS) and then converted to Cys-tRNA^Cys by Sep-tRNA:Cys-tRNA synthase (SepCysS). In this study, we searched all genomic and metagenomic protein sequence data in the Integrated Microbial Genomes (IMG) system and at the NCBI to reveal new clades of SepRS and SepCysS proteins belonging to diverse archaea in the four major groups (DPANN, Euryarchaeota, TACK, and Asgard) and two groups of bacteria ("Candidatus Parcubacteria" and Chloroflexi). Bacterial SepRS and SepCysS charged bacterial tRNA^Cys species with cysteine in vitro Homologs of SepCysE, a scaffold protein facilitating SepRS⋅SepCysS complex assembly in Euryarchaeota class I methanogens, are found in a few groups of TACK and Asgard archaea, whereas the C-terminally truncated homologs exist fused or genetically coupled with diverse SepCysS species. Investigation of the selenocysteine (Sec)- and pyrrolysine (Pyl)-utilizing traits in SepRS-utilizing archaea and bacteria revealed that the archaea carrying full-length SepCysE employ Sec and that SepRS is often found in Pyl-utilizing archaea and Chloroflexi bacteria. We discuss possible contributions of the SepRS-SepCysS system for sulfur assimilation, methanogenesis, and other metabolic processes requiring large amounts of iron-sulfur enzymes or Pyl-containing enzymes.IMPORTANCE Comprehensive analyses of all genomic and metagenomic protein sequence data in public databases revealed the distribution and evolution of an alternative cysteine-encoding system in diverse archaea and bacteria. The finding that the SepRS-SepCysS-SepCysE- and the selenocysteine-encoding systems are shared by the Euryarchaeota class I methanogens, the Crenarchaeota AK8/W8A-19 group, and an Asgard archaeon suggests that ancient archaea may have used both systems. In contrast, bacteria may have obtained the SepRS-SepCysS system from archaea. The SepRS-SepCysS system sometimes coexists with a pyrrolysine-encoding system in both archaea and bacteria. Our results provide additional bioinformatic evidence for the contribution of the SepRS-SepCysS system for sulfur assimilation and diverse metabolisms which require vast amounts of iron-sulfur enzymes and proteins. Among these biological activities, methanogenesis, methylamine metabolism, and organohalide respiration may have local and global effects on earth. Taken together, uncultured bacteria and archaea provide an expanded record of the evolution of the genetic code.

Keywords: biochemistry; bioinformatics; cysteine biosynthesis; genetic code; translation.

FIG 1

Distribution of SepRS, SepCysS, and SepCysE homologs in the prokaryotic domains of life. The bootstrap values (percentages) are shown for the unrooted maximum likelihood trees made with 100 replicates using MEGA 7. (A) Distribution of SepRS in archaea and bacteria. The archaeal species are (i) Rice cluster II or “Ca. Methanoflorentaceae” archaea (26); (ii) Thaumarchaeota AK59 archaea similar to clone AK59 locus tag AY555832 (all accession numbers are from GenBank unless otherwise specified) and clone 24Earc79 locus tag JN605031 (48, 82); (iii) “Ca. Bathyarchaeota” archaea (24, 38); (iv) three subgroups of uncultured Crenarchaeota (83) each similar to pSL50 gene U63342, pJP 33 gene L25300, and pJP 41 gene L25301, probably including “Ca. Verstraetearchaeota” archaea (21); (v) pMC2A209 archaea (49) similar to clone IAN1-71 locus tag AB175574 and clone ARC_OTU_72 locus tag KP091046 (47, 84); (vi) Hadesarchaea and MSBL1 archaea (36, 37); (vii) clone AK8/W8A-19 archaea similar to clone W8A-19 locus tag KM221272 (46) and clone AK8 locus tag AY555814 (48) or clone ARC_OTU_92 locus tag KP091068 (47); (viii) archaeon Odin LCB_4, “Ca. Lokiarchaeota” archaea, and a few unknown species in the Asgard superphylum (9, 31, 33); (ix) a Crystal Geyser groundwater (“Ca. Woesearchaeota”) archaeon most similar to archaeon GW2011_AR9 (85); (x) “Ca. Micrarchaeota” (Eury4AB group) archaea most similar to clone C1AA1CA10 locus tag GU127467 (86, 87); (xi) “Ca. Altiarchaeales” archaea (88–91); (xii) Crystal Geyser groundwater archaea (91), one of which is similar to clone SCA130 locus tag EU735580 (92); (xiii) locus tag Z7ME43 or Theionarchaea archaea (93); (xiv) Arc 1 group or “Ca. Methanofastidiosa” archaea (27); and (xv) unknown groups of archaea. The SepRS-harboring bacterial species are the “Ca. Parcubacteria” DG_74_2 bin bacterium (39), a putative deltaproteobacterium (CG bacterium no. 3), and Chloroflexi (probably Dehalococcoides) bacteria (34). SepRS sequences were classified into three clades and a few orphans. A few representative genetic loci of clade I SepRS genes are shown below the tree. As indicated, modern Crenarchaeota, including Thermofilum pendens, lack SepRS. TRAX belongs to translin superfamily proteins. FADS and RFK denote FAD synthase and riboflavin kinase, respectively. (B) Distribution of SepCysS in the prokaryotic domains of life. SepCysS sequences were classified into seven clades. (C) Distribution of SepCysE in three groups of selenocysteine-encoding archaea. (D) Multiple-alignment analysis of SepCysE homologs based on the crystal structure of M. jannaschii SepCysE (PDB accession no. 3wkr). The SepCysSn peptide is either encoded as a split gene preceding the SepCysS gene or N-terminally fused with SepCysS (SepCysSN).

FIG 2

Bacterial SepRS and SepCysS and “Ca. Parcubacteria” tRNA^Cys species. (A) Modeling of the N37-recognizing motif of SepRS. The crystal structure of A. fulgidus SepRS⋅tRNA^Cys (PDB accession no. 2du3) was used for the modeling. Although G37 in the crystal structure is unmodified, N¹-methylation may create a van der Waals interaction between the methyl group and the side chain of Ile444, as indicated with spheres. In bacterial SepRS species, Ile444 is replaced by hydrophilic Asp in most cases, and the following helix is totally missing. (B) Modeling of the dimer structure of SepCysS. The crystal structure of A. fulgidus SepCysS1 (PDB accession no. 2e7j) was used for the modeling. In bacterial SepCysS species, a loop (amino acids 144 to 147) is missing, and there is no insertion between amino acids 234 and 235. (C) Bacterial SepRS activity in vitro. Time course for plateau aminoacylation obtained by monitoring the accumulation of phosphoseryl-[³²P]tRNA^Cys using thin-layer chromatograms in Fig. S5A. tRNA^Cys substrates from the “Ca. Parcubacteria” DG_74_2 bin are indicated (G37 containing tRNA^Cys, [tRNA^Cys_arch], as a circle, its variant [tRNA^Cys_arch^G37A] as a triangle, and “Ca. Parcubacteria”-type tRNA^Cys as rectangles [tRNA^Cys_bac]). (D) Thin-layer chromatograms showing SepCysS-dependent O-phosphoseryl- to cysteinyl-[³²P]tRNA^Cys conversion. Reaction mixtures contained either “Ca. Parcubacteria” or Chloroflexi SepCysS (marked above the chromatogram). As a control, reaction mixtures containing only SepRS were also inspected (denoted “No SepCysS”). tRNA substrates are indicated below the chromatograms. Reaction products (O-phosphoseryl- and cysteinyl-[³²P]tRNA^Cys) were monitored as O-phosphoseryl- and cysteinyl-adenylates (Sep-AMP and Cys-AMP, respectively) after P1 nuclease digestion.

FIG 3

Distribution of PylRS in the prokaryotic domains of life. The bootstrap values (percentages) are shown for the unrooted maximum likelihood tree made with 100 replicates using MEGA 7. Bacterial origin is indicated with brown letters, while archaeal origin is indicated with black letters. Purple letters indicate a pending phylogenetic inference. Metagenomic origins are described in black parentheses, whereas “(pylSn)” indicates the existence of a pylSn gene. The occurrence of SepRS and Pyl-containing methyltransferases are shown next to the tree. For some of the archaeal bins in the hot spring metagenomes, the presence and absence of SepRS is pending, which is designated with “?.”